BIOMIMETIC SUPERSTRUCTURES FROM AMPHIPHILIC ABA - TRIBLOCK COPOLYMERS Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophich-Naturwissenschaftlichen Fakultät der Universität Basel von Julie GRUMELARD aus Saint-Louis, Frankreich BASEL, 2004 Genehmigt von der Philosophich-Naturwissenschaftlichen Fakultät Auf Antrag der Herren Prof. Dr. Wolfgang Meier (Universität Basel) Prof. Dr. Marcus Textor (ETH Zurich) Prof. Dr. Hanspeter Huber (Universität Basel) Basel, den 21.Oktober 2004 Prof. Dr. Hans-Jakob Wirz Dekan 2 « La science restera toujours la satisfaction du plus haut désir de notre nature, la curiosité; elle fournira à l'homme le seul moyen qu'il ait pour améliorer son sort. Elle préserve de l'erreur plutôt qu'elle ne donne la vérité; mais c'est déjà quelque chose d'être sûr de n'être pas dupe. » Ernest RENAN, L'Avenir de la science, Pensées de 1848 (1890), Flammarion 1995 3 Table of contents 1 ABSTRACT 6 2 ABBREVIATIONS 8 3 INTRODUCTION 9 BIOLOGICAL MEMBRANES 9 SYNTHETIC AMPHIPHILES AS BIOMEMBRANE MIMICS 10 PMOXA-PDMS-PMOXA ABA-TRIBLOCK COPOLYMERS 11 AIM OF THE PHD THESIS 14 4 NANOVESICLES 16 4.1 ETHANOL METHOD 16 4.2 DETERGENT METHOD 32 4.2.1 DIALYSIS 34 4.2.2 DILUTION METHOD 36 4.2.3 BIO-BEADS METHOD 38 4.2.4 MEMBRANE PROTEIN RECONSTITUTION 45 4.3 BULK SWELLING 50 4.4 REMINDER OF MAIN RESULTS 53 5 NANOTUBES 54 5.1 BULK DISSOLUTION OR BULK SWELLING 54 5.2 FILM REHYDRATION OR FILM SWELLING 57 5.3 CRYO-TEM 65 5.4 FLUORIMETRY 70 5.5 GOLD LOADED NANOTUBES 72 5.5.1 GOLD PARTICLES ENCAPSULATION 72 5.5.2 GOLD TEMPLATED FORMATION 73 5.6 REMINDER OF MAIN RESULTS 81 6 CONCLUSION AND OUTLOOKS 82 7 MATERIAL AND METHODS 84 7.1 POLYMERS 84 7.2 COMPLEX I RECONSTITUTION EXPERIMENTS 85 7.3 ETHANOL METHOD 86 7.4 DETERGENT REMOVAL WITH BIO-BEADS 86 7.5 ATOMIC FORCE MICROSCOPY 87 7.6 DIALYSIS 87 7.6.1 DIALYSIS MEMBRANES 87 4 7.6.2 TEMPERATURE-CONTROLLED, CONTINUOUS OPEN-FLOW DIALYSIS APPARATUS87 7.7 DILUTION METHOD 88 7.7.1 THE DILUTION CELL WITH OPTICAL DETECTOR 88 7.7.2 CONTROL UNIT 89 7.8 DETERMINATION OF THE FREE DETERGENT CONCENTRATION USING THE SITTING-DROP METHOD 90 7.9 TRANSMISSION ELECTRONIC MICROSCOPY 91 7.9.1 PREPARATION OF CARBON-PARLODION* COMPOSITE POLYMER FILMS ON COPPER GRIDS 91 7.9.2 NEGATIVE STAINING 91 7.9.3 CRYOELECTRON MICROSCOPY (CRYO-EM) 92 7.9.4 FREEZE FRACTURE PREPARATIONS FOR ELECTRON MICROSCOPY. 92 7.10 DYNAMIC LIGHT SCATTERING 92 7.11 FLUORESCENCE MEASUREMENTS 93 7.11.1 FLUORESCENCE MICROSCOPY 93 7.11.2 FLUORIMETRY 93 8 APPENDIX I: OTHER ABA-TRIBLOCK COPOLYMER SUPERSTRUCTURES 94 8.1 GIANT VESICLES 94 8.2 MONOLAYERS 95 9 APPENDIX II: FREE-STANDING FILMS – BILAYERS 106 9.1 INTRODUCTION 106 9.2 EXPERIMENTAL 106 9.3 RESULTS 108 9.4 CONCLUSION AND PERSPECTIVES 110 10 APPENDIX III: POLYMER NANOCONTAINERS FOR SELECTIVE IMMOBILIZATION AT SURFACES 111 11 PUBLICATIONS AND POSTERS 113 POSTER PRESENTATIONS 113 PUBLICATION LIST 113 12 ACKNOWLEDGMENTS 116 13 REFRERENCES 118 5 1 Abstract The spontaneous formation of nanostructured materials by molecular self-assembly of block copolymers is an active area of research, driven both by its inherent beauty and by a wealth of potential technological applications. The so-called “supramolecular” structures can be used to build functional materials with nanoscopic dimensions, such as sensors for biochips or smart drug delivery vehicles. Block copolymer vesicles have attracted increasing interest, particularly in view of possible applications in drug delivery and in protein reconstitution. Conventional methods utilizing synthetic lipid membranes for protein functionality assays have yielded much information with respect to the membrane protein behavior. Furthermore, amphiphilic block copolymer membrane, allowing proper protein refolding while preserving protein function, have been developed to improve the efficiency of these proteins in robust devices. Even if spherical structures are still the most common supramolecular structures generated by self-assembly of block copolymers, a remarkable variety of other morphologies have now been demonstrated, such as rod-like micelles and nanotubes. Soft nanotubes made from biocompatible organic molecules and polymers could find applications in biotechnology and medicine. However, polymer hollow tubes are rare and have so far only been described in organic solvents where their fabrication often requires elaborate procedures. Recently, a series of ABA triblock copolymer composed of poly(dimethylsiloxane)- block- poly(2-methyloxazoline)-block- poly(dimethylsiloxane) (PMOXA-b-PDMS-b- PMOXA) able to mimic biomembranes has been introduced. The PMOXA blocks have hydroxyl end groups that allow functionalization with methacrylic acid. In aqueous solution, the triblock macromonomers form supramolecular assemblies that can be chemically cross-linked by polymerization of the methacrylic acid groups. Established and new preparation methods have been used to prepare superstructures in water with various hydrophobic-hydrophilic ratios of the PMOXA-b-PDMS-b- 6 PMOXA ABA-triblock copolymers. Suitable choice of the block lengths and preparation method allowed controlling the shape of the self-assemblies. We have particularly introduced a preparation method for nanovesicles using detergents and bio-beads that is a suitable alternative devoid of organic solvents leading to improved reconstitution of functional membrane proteins. We have also developed a simple method for the preparation of soft, water-filled nanotubes via self-assembly of PMOXA-b-PDMS-b-PMOXA in aqueous media. Polymer nanotubes have been loaded with water-soluble substances and used as highly specific templates for inorganic synthesis. 7 2 Abbreviations AFM Atomic Force Microscopy BLM Black Lipid Membrane CAC Critical aggregation concentration CF Carboxyfluorescein CMC Critical micellar concentration DDM Dodecylmaltoside or n-Dodecyl-β-D-maltopyranoside 2-D Two-dimensional DLS Dynamic Light Scattering DNA Deoxyribonucleic acid DPHPC Diphytanoylphosphatidylcholine E. coli Escherichia coli EPR Electron Paramagnetic Resonance HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, or “Free Acid” LamB Maltoporin MscL Large mechanosensitive channel NMR Nuclear Magnetic Resonance OG Octylglucoside or n-Octyl-β-D-glucopyranoside OmpF E. coli Outer membrane porin o-POE Octyl-POE or n-octyl polyoxyethylene PBS Phosphate Saline Buffer PDMS Poly (dimethylsiloxane) PEG Polyethylene glycol PMOXA Poly (2-methyloxazoline) SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEC Size Exclusion Chromatography TEM Transmission Electron Microscopy T X100 Triton X100 8 3 Introduction Biological membranes The function of biological membranes [1] is to organize biological processes by compartmentalizing them. Indeed, the cell, the basic unit of life, is essentially defined by its enveloping plasma membrane. Moreover, in eukaryotes, many subcellular organelles, such as nuclei, mitochondria, chloroplasts, the Golgi apparatus and the endoplasmic reticulum, are likewise membrane bound. Many fundamental biochemical processes, e.g. electron transport and oxidative phosphorylation, occur on or in the membrane scaffold. A biological membrane is composed of proteins and other molecules associated with a phospholipid bilayer matrix (Figure 1). The lipids are arranged tail to tail so that only the hydrophilic heads are exposed to the aqueous medium on both sides of the membrane. This is the minimum-energy configuration for a suspension of lipids in water. Figure 1: Schematic presentation of a cell membrane. Integral proteins (orange) are embedded in a bilayer of phospholipids (blue, shown in higher proportion than in nature for clarity) and cholesterol (yellow). The carbohydrate components of glycoproteins (yellow beaded chains) and glycolipids (green beaded chains) occur only on the external face of the membrane. From [1] p 292 9 Plasma membrane has various functions; (i) it acts as a semi-permeable barrier, (ii) it regulates the transport in and out of the cell, (iii) and it is responsible for communication and adhesion between the cells. The lipid bilayer accounts for the basic barrier functions of the plasma membrane. It is permeable to water, oxygen, small hydrophobic molecules such as steroids, and ethanol; it is impermeable to highly charged molecules and ions such as Na+, K+. Membrane proteins carry out most of the functions of the membrane. They are channels, receptors, enzymes etc., as shown in Figure 2. The functions of membrane proteins include transport of substances across membranes, enzymatic activity (e.g. smooth endoplasmic reticulum), signal transduction (e.g. cell communication), intracellular joining, cell-cell recognition (e.g. cell communication), and attachment to the cytoskeleton and extracellular matrix. Figure 2: Some functions of plasma membrane proteins 1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter http://www.essentialcellbiology.com Published by Garland Publishing. Synthetic amphiphiles as biomembrane mimics Artificial lipid membranes are widely studied as a model system for biological membranes. They are useful to study the physical properties of membranes, such as elasticity and permeability, but also the structure and the function of membrane proteins; their functional insertion and moreover their crystallization in lipid bilayers is of a great interest. In addition, liposomes (closed, self-sealing, solvent-filled vesicles surrounded by a single lipid bilayer) have attracted considerable interest for drug delivery [2],